Project Summary/Abstract Neurotransmitter release is influenced by drugs, and certain mental disorders such as depression, but the details are unclear. It is mediated by the SNARE complex, complexin (Cpx), synaptotagmin (Syt), and other proteins. Asynchronous release is a mode of neurotransmitter release. It has received increasing attention as it may generate or compensate abnormal neural activities, and play critical role in pre-synaptic plasticity. While most neuronal communication relies upon synchronous release triggered by the Ca2+ sensors Syt1, 2 and 9 (collectively called ?fast Syts?). Asynchronous release has a longer, variable delay after an action potential or series of action potentials, and it is mediated by the Ca2+ sensor Syt7. Fast Syts and Syt7 share a similar domain structure and a high degree of homology in their Ca2+-binding C2A and C2B domains. Recently I have identified the primary SNARE-Syt1 interface between Syt1 C2B domain and the SNARE complex, revealing the primary interface is specific for synchronous release. In contrast, Syt7 does not form a similar primary interface. These results raise the following questions: 1) how does Syt7's cooperate with SNAREs and Cpx to trigger delayed asynchronous release; 2) what is the difference between fast Syts and Syt7 that gives rise to the different modes of neurotransmitter release; 3) how is asynchronous release regulated by Syts and Ca2+. The overall goal of this proposal is the elucidation of the molecular mechanism and regulation of asynchronous release. The proposed study will examine how Syt7 mediates asynchronous release in cultured cortical neurons and brain slices from Syt1/7 double knockout mice with re-introduction of mutates designed based on sequence analysis and a newly solved crystal structure of SNARE-Cpx-Syt1 complex. Finally, I will reveal the interaction among Syt1/7, Cpx, SNARE complex and membrane, and investigate the regulation of asynchronous release by Syt1, Syt7 and Ca2+. Combined with my work on synchronous release, such results are expected to provide a better understanding of the roles of neurotransmitter release in neural activity and mental disorders, may lead to new therapeutics for the prevention and treatment of a variety of mental disorders. In addition, the results are also expected to vertically advance the understanding of pre-synaptic plasticity which is believed to be related to memory, learning, and behavior, as well as fundamentally advance the field of neuroscience. My long-term career goal is to lead a world-class laboratory in the forefront of synaptic physiology. I intend to use techniques in structural biology, single molecule spectroscopy and imaging, as well as key techniques in neuroscience as investigation tools. To complement my knowledge of structural biology acquired during my PhD, I will carry out the mentored phase of this Award as a postdoctoral Research Fellow in the laboratories of Drs. Axel Brunger and Thomas Sdhof. I have designed an ambitious research career development plan to achieve my immediate goals for the mentored period: 1) expand my advanced scientific and technical knowledge, and 2) prepare my transition to independence. Under the mentorship of Drs. Brunger and Sdhof, I will follow a structured training program to enhance my professional abilities to establish and run my own laboratory. Stanford Medical School will provide me with ideal environment to fully benefit from this Award and become a successful independent scientist.